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In natural populations of Barbarea vulgaris we found two distinctly different glucosinolate profiles. The most common glucosinolate profile is dominated (94%) by the hydroxylated form, (S)-2-hydroxy-2-phenylethyl-glucosinolate (glucobarbarin, BAR-type), whereas in the other type 2-phenylethyl-glucosinolate (gluconasturtiin, NAS-type) was most prominent (82%). NAS-type plants have a 108-fold increase of gluconasturtiin concentration in rosette leaves compared to BAR-type plants. The glucosinolate composition of both chemotypes is consistent throughout all plant organs and after induction with jasmonic acid. Although the glucosinolate profile of the roots has a more diverse composition than other plant organs, it still matches the chemotype. In 12 natural populations that we sampled in Germany, Belgium, France and Switzerland solely BAR-type plants were found. However, eight out of the 15 Dutch populations that were sampled contained 2–22% NAS-type plants. Controlled crosses showed that the chemotype was heritable and determined by a single gene with two alleles. The allele coding for the BAR-type was dominant and the allele for the NAS-type was recessive. The different glucosinolate profiles will yield different hydrolysis products upon damage, and therefore we expect them to differentially affect the multitrophic interactions associated with B. vulgaris in their natural environment. [KEYWORDS: Barbarea vulgaris ; Brassicaceae ; Glucosinolates ; Glucobarbarin ; Gluconasturtiin ; Plant defence ; Polymorphism; Heritability]